BRD4 Degradation By Protacs Represents a More Effective Therapeutic Strategy Than BRD4 Inhibitors in DLBCL

Introduction: BRD4, a member of the bromodomain and extraterminal domain (BET) family, has emerged as an attractive target in multiple oncology indications, particularly hematological malignancies. As a crucial chromatin reader, BRD4 is frequently found to be preferentially localized at super-enhancer regions, which often reside upstream of important oncogenes, including c-Myc, Bcl-xL and Bcl-6. Thus BRD4 inhibitors have gained attention as promising therapeutics in malignancies driven by these key oncogenes, including Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL). However, we have recently found that BRD4 inhibitors lead to a compensatory accumulation of BRD4, which results in an incomplete inhibition of c-MYC and lack of apoptosis. These limitations could severely hamper the therapeutic potential of BRD4 inhibitors. To circumvent these limitations, we designed BRD4 degraders using our PROTAC (PROteolysis TArgeting Chimera) platform (Lu et al. Chem Biol. 22:755-763, 2015) and investigated the effects of BRD4 degradation in DLBCL in comparison with inhibitors.

Methods and Results: We designed specific BRD4 PROTACs, which contain a BRD4 binding moiety connected to an E3 ubiquitin ligase-recruiting moiety.  In all ABC-DLBCL, and GCB-DLBCL cell lines tested, BRD4 PROTACs lead to potent (<10 nM), fast (<4hrs) and robust (>90%) degradation of BRD4. This degradation is accompanied by more pronounced and longer-lasting suppression of downstream signaling events, such as c-MYC, than small molecule inhibitors (JQ1 and OTX015) treatment. We confirmed that BRD4 degradation is E3 ligase-mediated and proteasome-dependent as both excessive E3 ligase binding ligand and proteasome inhibitors (MG132 and carfilzomib) were able to prevent PROTAC-induced BRD4 degradation. Most importantly, BRD4 PROTACs were more effective in inhibiting DLBCL cell proliferation (measured by CellTiter-Glo® Luminescent Cell Viability Assay) and inducing apoptosis (evaluated by caspase3/7 assay and PARP cleavage) compared to BRD4 inhibitors.  

Conclusion: Hijacking E3 ubiquitin ligases, through PROTAC technology, is an innovative approach to achieve targeted, specific protein degradation. Such targeted degradation can provide important therapeutic advantages. Here, we demonstrated that BRD4 PROTACs lead to robust BRD4 downregulation, which results in better downstream signaling suppression, more pronounced inhibition of proliferation and induction of apoptosis compared to the effects of small molecule inhibitors. The results of on-going efficacy studies using a DLBCL tumor xenograft model will be presented at the meeting. Taken together, BRD4 degradation by PROTACs is a promising therapeutic strategy in DLBCL.